The mechanisms of action and regulation of heart mitochondrial malate dehydrogenase are being studied, with particular emphasis on possible allosteric effects. These effects are being elucidated by fluorescence techniques and steady-state and transient kinetics. Anomalous kinetic features and the effects of various modifiers on the reaction mechanism are being studied in detail, with particular emphasis on transient intermediates. Subunit dissociation and conformational changes due to ligand binding will be evaluated using fluorescence energy transfer and fluorescence polarization. It is anticipated that this project will provide further information regarding the reaction mechanism of malate dehydrogenase as well as an additional understanding of the general phenomenon of allosteric regulation of enzymes.